Comparative Colorimetric Sensor Based on Bi-Phase γ-/α-Fe2O3 and γ-/α-Fe2O3/ZnO Nanoparticles for Lactate Detection.

This work reports on Fe2O3 and ZnO materials for lactate quantification. In the synthesis, the bi-phase γ-/α-Fe2O3 and γ-/α-Fe2O3/ZnO nanoparticles (NPs) were obtained for their application in a lactate colorimetric sensor. The crystalline phases of the NPs were analyzed by XRD and XPS techniques. S/TEM images showed spheres with an 18 nm average and a needle length from 125 to 330 nm and 18 nm in diameter. The γ-/α-Fe2O3 and γ-/α-Fe2O3/ZnO were used to evaluate the catalytic activity of peroxidase with the substrate 3,3,5,5-tetramethylbenzidine (TMB), obtaining a linear range of 50 to 1000 µM for both NPs, and a 4.3 µM and 9.4 µM limit of detection (LOD), respectively. Moreover, γ-/α-Fe2O3 and γ-/α-Fe2O3/ZnO/lactate oxidase with TMB assays in the presence of lactate showed a linear range of 50 to 1000 µM, and both NPs proved to be highly selective in the presence of interferents. Finally, a sample of human serum was also tested, and the results were compared with a commercial lactometer. The use of ZnO with Fe2O3 achieved a greater response toward lactate oxidation reaction, and has implementation in a lactate colorimetric sensor using materials that are economically accessible and easy to synthesize.

Elucidating the Mechanism behind the Bionanomanufacturing of Gold Nanoparticles Using Bacillus subtilis.

Over the last two decades, gold nanoparticles (GNPs) have opened up numerous research and industrial opportunities in biomedical, optical, and electronic fields due to their size- and morphology-dependent properties [Grassian, V. H. Macromolecules 2008, 112(47), 18303-18313 and Nehl, C. L.; Hafner, J. H. J. Mater. Chem. 2008, 18(21), 2415-2419]. Therefore, green and efficient synthesis strategies providing precise control over size and morphology are desired. Since biological catalysts are known for the selectivity, efficiency, and environmentally friendly production of gold nanoparticles (referred to as bionanomanufacturing), they have been considered for GNP synthesis. However, the mechanism of how most of these biological entities produce GNPs has not been elucidated to date, limiting the industrial implementation of complex biological systems for nanoparticle synthesis. In this study, we investigated the mechanism of extracellular GNP production by Bacillus subtilis (B. subtilis). It is shown that B. subtilis releases vegetative catalase (Cat A) into the supernatant. Cat A from the supernatant and commercial catalase were employed to establish the mechanism of GNP formation. The bionanomanufactured GNPs were characterized using ultraviolet-visible (UV-vis) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Based on our results, we theorize that the mechanism of extracellular GNP production by B. subtilis Cat A involves (1) formation of gold-thiol bonds followed by (2) stabilization of GNPs with the denatured bacterial protein that serves as a capping agent. This research offers early insights into the gold-reducing mechanism occurring in the cell-free extract of B. subtilis, which can potentially lead to the design of protocols for the controlled production of GNPs with isolated enzymes at the industrial scale.

Multimerization of an Alcohol Dehydrogenase by Fusion to a Designed Self-Assembling Protein Results in Enhanced Bioelectrocatalytic Operational Stability.

Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.

Quantitative Framework for Stochastic Nanopore Sensors Using Multiple Channels.

Membrane protein channels employed as stochastic sensors offer large signal-to-noise ratios and high specificity in single molecule binding measurements. Stochastic events in a single ion channel system can be measured using current-time traces, which are straightforward to analyze. Signals arising from measurement using multiple ion channels are more complicated to interpret. We show that multiple independent ion channels offer improved detection sensitivity compared to single channel measurements and that increased signal complexity can be accounted for using binding event frequency. More specifically, the leading edge of binding events follows a Poisson point process, which means signals from multiple channels can be superimposed and the association times (between each binding event leading edge), allow for sensitive and quantitative measurements. We expand our calibration to high ligand concentrations and high numbers of ion channels to demonstrate that there is an upper limit of quantification, defined by the time resolution of the measurement. The upper limit is a combination of the instrumental time resolution and the dissociation time of a ligand and protein which limits the number of detectable events. This upper limit also allows us to predict, in general, the measurement requirements needed to observe any process as a Poisson point process. The nanopore-based sensing analysis has wide implications for stochastic sensing platforms that operate using multiple simultaneous superimposable signals.

Enzymatic Electrosynthesis of Alkanes by Bioelectrocatalytic Decarbonylation of Fatty Aldehydes.

An enzymatic electrosynthesis system was created by combining an aldehyde deformylating oxygenase (ADO) from cyanobacteria that catalyzes the decarbonylation of fatty aldehydes to alkanes and formic acid with an electrochemical interface. This system is able to produce a range of alkanes (octane to propane) from aldehydes and alcohols. The combination of this bioelectrochemical system with a hydrogenase bioanode yields a H2 /heptanal enzymatic fuel cell (EFC) able to simultaneously generate electrical energy with a maximum current density of 25 µA cm-2 at 0.6 V and produce hexane with a faradaic efficiency of 24 %.

Polymer-immobilized, hybrid multi-catalyst architecture for enhanced electrochemical oxidation of glycerol.

The development of a hybrid, tri-catalytic architecture is demonstrated by immobilizing MWCNTs, TEMPO-modified linear poly(ethylenimine) and oxalate decarboxylase on an electrode to enable enhanced electrochemical oxidation of glycerol. This immobilized, hybrid catalytic motif results in a synergistic 3.3-fold enhancement of glycerol oxidation and collects up to 14 electrons per molecule of glycerol.

Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors.

The development of structure-switching, electrochemical, aptamer-based sensors over the past ∼10 years has led to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quantitative characterization of the analytical performance of electrochemical aptamer-based sensors. Although the limits of detection of reported electrochemical aptamer-based sensors do not often reach that of gold-standard methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting analytical applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss the challenges and prospects for this class of sensors.

Bioinspired Protein Channel-Based Scanning Ion Conductance Microscopy (Bio-SICM) for Simultaneous Conductance and Specific Molecular Imaging.

The utility of stochastic single-molecule detection using protein nanopores has found widespread application in bioanalytical sensing as a result of the inherent signal amplification of the resistive pulse method. Integration of protein nanopores with high-resolution scanning ion conductance microscopy (SICM) extends the utility of SICM by enabling selective chemical imaging of specific target molecules, while simultaneously providing topographical information about the net ion flux through a pore under a concentration gradient. In this study, we describe the development of a bioinspired scanning ion conductance microscopy (bio-SICM) approach that couples the imaging ability of SICM with the sensitivity and chemical selectivity of protein channels to perform simultaneous pore imaging and specific molecule mapping. To establish the framework of the bio-SICM platform, we utilize the well-studied protein channel α-hemolysin (αHL) to map the presence of ß-cyclodextrin (ßCD) at a substrate pore opening. We demonstrate concurrent pore and specific molecule imaging by raster scanning an αHL-based probe over a glass membrane containing a single 25-µm-diameter glass pore while recording the lateral positions of the probe and channel activity via ionic current. We use the average channel current to create a conductance image and the raw current-time traces to determine spatial localization of ßCD. With further optimization, we believe that the bio-SICM platform will provide a powerful analytical methodology that is generalizable, and thus offers significant utility in a myriad of bioanalytical applications.

Monitoring cooperative binding using electrochemical DNA-based sensors.

Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3' terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM-4 µM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein-DNA binding mechanisms.

Monitoring charge flux to quantify unusual ligand-induced ion channel activity for use in biological nanopore-based sensors.

The utility of biological nanopores for the development of sensors has become a growing area of interest in analytical chemistry. Their emerging use in chemical analysis is a result of several ideal characteristics. First, they provide reproducible control over nanoscale pore sizes with an atomic level of precision. Second, they are amenable to resistive-pulse type measurement systems when embedded into an artificial lipid bilayer. A single binding event causes a change in the flow of millions of ions across the membrane per second that is readily measured as a change in current with excellent signal-to-noise ratio. To date, ion channel-based biosensors have been limited to well-behaved proteins. Most demonstrations of using ion channels as sensors have been limited to proteins that remain in the open, conducting state, unless occupied by an analyte of interest. Furthermore, these proteins are nonspecific, requiring chemical, biochemical, or genetic manipulations to impart chemical specificity. Here, we report on the use of the pore-forming abilities of heat shock cognate 70 (Hsc70) to quantify a specific analyte. Hsc70 reconstitutes into phospholipid membranes and opens to form multiple conductance states specifically in the presence of ATP. We introduce the measurement of "charge flux" to characterize the ATP-regulated multiconductance nature of Hsc70, which enables sensitive quantification of ATP (100 µM-4 mM). We believe that monitoring protein-induced charge flux across a bilayer membrane represents a universal method for quantitatively monitoring ion-channel activity. This measurement has the potential to broaden the library of usable proteins in the development of nanopore-based biosensors.